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ssDNA 100-R Kit - SCIEX

ssDNA 100-R Kit Care and Use Instructions 726479AE. July 2010. Beckman Coulter, Inc. 250 S. Kraemer Blvd., Brea, CA 92821. Copyright 2010 Beckman Coulter, Inc. Copyright, Licenses and Trademarks Copyright Beckman Coulter, Inc., 2010. All rights reserved. No part of this publication may be reproduced, transcribed, transmitted, or translated into any language in any form by any means without the written permission of Beckman Coulter, Inc. Licenses and Trademarks Beckman Coulter is a registered trademark of Beckman Coulter, Inc. Table of Contents Introduction .. 1-1. Kit Contents (Kit Reorder Number 477480).

ssDNA 100-R Kit 726479AE 1-1 1 ssDNA 100-R Kit Introduction The Beckman Coulter ssDNA 100-R Kit provides rapid separation and analysis of oligonucleotides having 10 to 100 bases of length.

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Transcription of ssDNA 100-R Kit - SCIEX

1 ssDNA 100-R Kit Care and Use Instructions 726479AE. July 2010. Beckman Coulter, Inc. 250 S. Kraemer Blvd., Brea, CA 92821. Copyright 2010 Beckman Coulter, Inc. Copyright, Licenses and Trademarks Copyright Beckman Coulter, Inc., 2010. All rights reserved. No part of this publication may be reproduced, transcribed, transmitted, or translated into any language in any form by any means without the written permission of Beckman Coulter, Inc. Licenses and Trademarks Beckman Coulter is a registered trademark of Beckman Coulter, Inc. Table of Contents Introduction .. 1-1. Kit Contents (Kit Reorder Number 477480).

2 1-1. Materials Required but Not Provided .. 1-2. Preparing the Tris-Borate-Urea Buffer .. 1-3. Using the Refrigerated Tris-Borate-Urea Buffer .. 1-4. Preparing ssDNA 100-R Gel .. 1-5. For 1 to 8 runs: .. 1-5. For Multiple Runs (more than 8): .. 1-5. Installing the Coated Capillary .. 1-6. ssDNA 100-R Method .. 1-6. Preparing the Test Mix .. 1-11. Preparing the Oligonucleotide Sample .. 1-11. Performing a Test Run .. 1-11. Important Notes .. 1-13. Checking the Results .. 1-13. Troubleshooting Guide .. 1-15. Additional Information .. 1-16. Other Beckman Coulter CE Chemistry Kits: .. 1-16. Other Beckman Coulter Literature.

3 1-17. Additional Technical Support .. 1-17. North America: .. 1-17. Worldwide: .. 1-17. iii iv 1ssDNA 100-R Kit Introduction The Beckman Coulter ssDNA 100-R Kit provides rapid separation and analysis of oligonucleotides having 10 to 100 bases of length. This kit features a replaceable gel buffer with a coated capillary for maximum reproducibility. This kit is not recommended for use with a photodiode array detector (PDA). This kit should be used with a UV Detector equipped with a 254 nm filter. Kit Contents (Kit Reorder Number 477480). Product Name Quantity/Amount DNA Capillary, 65 cm, 100 m 2. ssDNA 100-R Gel g (lyophilized).

4 Tris-Borate Buffer 1 bottle 7 M Urea 1 bottle Test Mix pd(A) 40-60 ssDNA 100-R Care and Use Instructions 1. Upon receipt: r store the DNA Capillary and lyophilized gel at 2-8 C. r store Test Mix at -20 C. r store unreconstituted Tris-borate and urea buffer bottles at room temperature ssDNA 100-R Kit 726479AE 1-1. The following items may be reordered separately from the Kit: Product Name Part Number DNA Capillary 477477. ssDNA 100-R Gel 477621. pd(A) 40-60 Test Mix 477626. ssDNA 100 Buffer Kit 338481. (Tris-Borate, Urea). Materials Required but Not Provided r Deionized Water r Micropipet to deliver 500 L.

5 R Capillary Cartridge, blank, 100 x 200 m aperture (PN 144738). r PCR vials, 200 L (PN 144709). r Micro vial springs (PN 358821). r 2 mL vials (PN 144980). r 2 mL vial cap, red (PN 144648). r PCR vial cap, gray (PN 144656). r PCR vial holder (PN 144657). r vortex mixer r magnetic stir plate with stir bar r 10 mL disposable syringes r m membrane syringe filter r m membrane syringe filter ssDNA 100-R Kit 1-2. Preparing the Tris-Borate-Urea Buffer 1. Add 135 mL of 16 to 18 megohm deionized water to the bottle containing the dry tris-borate buffer. 2. To dissolved the buffer, stir for 20 to 30 minutes using a stirring bar that has been cleaned with methanol and rinsed with deionized water.

6 Be sure that the boric acid is completely dissolved before proceeding to the next step. 3. Slowly add the dry 7M urea to the bottle of dissolved tris-borate buffer while using the magnetic stir bar to mix the solution. The dissolution of urea is endothermic, so the bottle will get very cold. After approximately two hours of stirring, the buffer solution should be clear. The Tris-Borate-Urea buffer is now ready for use. CAUTION Do not heat the buffer solution to speed the warming process. This will shorten the usable life of the buffer. 4. Remove the required volume for the day's use and filter through m filter.

7 Store the remainder at 2 C to 8 C. NOTE The usable life of the reconstituted buffer is 30 days when stored at 2 C to 8 C. Some magnetic stirrers produce enough heat to degrade the urea. A small piece of corrugated cardboard can be used as an insulator between the buffer and the stirrer to minimize the heating. ssDNA 100-R Kit 726479AE 1-3. Using the Refrigerated Tris-Borate-Urea Buffer 1. If the buffer solution was previously reconstituted and refrigerated, bring the entire container of buffer to ambient temperature before use while stirring slowly and continuously with a clean stirring bar. 2. Remove the required volume to be used for the day and filter through a m disposable syringe filter into a clean container.

8 3. Pipet mL of Tris-Borate-Urea buffer into each of the required 2 mL. vials. 4. Seal the vials with the red caps. 5. Sonicate for 5 minutes to degas the buffer solution. The following points are important when using refrigerated buffer solution: r For optimal migration time reproducibility, replace the vials of tris-borate-urea buffer after 18 runs. r The current should be monitored at all times. Ionic strength changes, gel degradation, and/or the formation of bubbles are indicated by change in the average current value, or current fluctuations. r Use only Beckman Coulter Tris-Borate buffer and 7M Urea with the Beckman Coulter DNA Capillary.

9 The purity of the buffer raw material components is critical to the life of the coated capillary and of the buffer. Do not substitute buffer components from other vendors. ssDNA 100-R Kit 1-4. Preparing ssDNA 100-R Gel 1. Add mL of filtered tris-borate-urea buffer to the lyophilized gel. 2. Use a clean magnetic stir bar to stir the solution until the gel is completely dissolved (4 to 6 hours). For 1 to 8 runs: 1. Transfer 200 L of the filtered gel into a PCR vial. 2. Centrifuge (6,000 rpm maximum) the vial for no more than 2 minutes to remove air bubbles. 3. Place the PCR vial in a vial holder equipped with a spring and seal it with a gray cap.

10 For Multiple Runs (more than 8): 1. Filter the gel through a m disposable syringe filter and pipet mL into a 2 mL vial. 2. Seal the vial with a red cap and sonicate the vial 5 times for 30 seconds, each time. 3. Allow the air bubbles to rise to the surface between each sonicating cycle. The following points are important when using ssDNA 100-R gel: r On-board stability for a 2 mL vial filled with gel is 24 hours. r If using 200 L of gel, do not leave the gel in the sample tray for more than 5 hours. This may result in an increased migration time due to an increase in the viscosity of gel. r For optimal migration time reproducibility, replace the gel in the capillary after every 8 runs.


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